IMPROVING DISEASE RESISTANCE OF LIVESTOCK THROUGH IDENTIFYING MECHANISMS OF VITAMIN D SIGNALING IN THE IMMUNE SYSTEM

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1002526
• Project Start Date: Feb 20, 2014
• Project End Date: Dec 31, 2018
• Program Code: [(N/A)]- (N/A)

Project Director
Nelson, CO, D.

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
Animal Sciences

Non Technical Summary
Infectious diseases are estimated to cost the livestock industries several billions of dollars in lost revenue every year. The goal of this project is to decrease the economic and food safety costs of those diseases through improving disease resistance of animals. Vitamin D deficiency is correlated with increased risk for infectious diseases in humans, the same is likely true for cattle and pigs. This project will identify host and pathogen factrs that affect vitamin D metabolism in cattle and pigs, and the influence of vitamin D compounds on immunity of cattle and swine. This information gained from this project is expected to generate strategies, therapies, and nutritional recommendations that will improve disease resistance of livestock.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
305	3410	1090	50%
305	3510	1090	50%

Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
3510 - Swine, live animal; 3410 - Dairy cattle, live animal;

Field Of Science
1090 - Immunology;

Keywords

immunity

vitamin d

nutrition

livestock

Goals / Objectives
The goal of this project is to decrease the impact of infectious diseases in livestock (e.g. mastitis, tuberculosis, respiratory infection) through improving disease resistance of livestock.Objectives:1. Identify effects of pathogen and host factors on vitamin D metabolism in the immune system. The active vitamin D metabolite influences several key immune responses, and the magnitude of that signal is dictated by the rate at which vitamin D is metabolized in the immune cells. This objective will investigate the effects of pathogen-associated factors, host cytokines, and host genetic factors on CYP27B1 (vitamin D activating enzyme) and CYP24A1 (deactivating enzyme) abundance in immune cells of swine and cattle. This objective is expected to identify the factors that promote the vitamin D pathway and factors that inhibit the vitamin D pathway.2. Determine the influence of vitamin D signaling on bovine and porcine immune responses. The effects of vitamin D on immunity is barely understood. This objective will identify the genes that are regulated by the vitamin D pathway in immune cells, along with the physiological impact of inluencing those genes through the vitamin D pathway (e.g. antimicrobial activity, phagocytosis, inflammation). This objective also will evaluate the impact of vitamin D status, the concentration of vitamin D metabolites in the body, on the ability of pathogens to infect or survive in the host animal.

Project Methods
Objective 1: Identify effects of pathogen factors on vitamin D metabolism in bovine and porcine immune cells. Lipopolysaccharide (LPS) activates a vitamin D signaling pathway in in bovine macrophages and preliminary data indicates the same for swine. Several experiments to follow-up the preliminary data are planned to study the influence of pathogen associated molecules on vitamin D metabolism in vitro and in vivo (experiments 1 and 2).Experiment 1.1: Evaluate the influence of TLR signaling on vitamin D metabolism in macrophages. Monocytes from peripheral blood of pigs and cattle will be evaluated for CYP27B1 and VDR gene and protein expression in response to activation with TLR ligands (dsRNA, lipolysaccharide, lipoteichoic acid, peptidoglycan, and triacylated lipopeptides). The ability of the pig monocytes to synthesize 1,25(OH)2D3 will be accomplished by addition of physiological concentrations of 25(OH)D3 (25-100 ng/mL) to resting and TLR-stimulated monocytes, followed by analysis of CYP24A1 gene expression (and genes identified from experiment 2). Upregulation of CYP24A1 gene expression in response to TLR-stimulation and addition of 25(OH)D3 will indicate that TLR-stimulated monocytes are produce 1,25(OH)2D3. A treatment with an inhibitor of CYP27B1, ketoconazole, also will be included to demonstrate that effects of 25(OH)D3 in TLR-stimulated monocytes are dependent on CYP27B1 enzyme activity. Alternatively, CYP27B1 expression will be inhibited with siRNA specific for the CYP27B1 transcript to determine the requirement of the CYP27B1 enzyme. The actual amount of 1,25(OH)2D3 produced by monocytes as a result of LPS and 25(OH)D3 treatments would be an ideal measure, but there are currently not reliable assays to directly measure 1,25(OH)2D3 in cell culture because of metabolites produced in culture that interfere with the assay.Experiment 1.2: Determine if the vitamin D pathway is activated in immune cells in vivo as a result of influenza infection. Lung tissue samples, alveolar macrophages, and draining lymph nodes will be collected from necropsies of H1N1-infected and control pigs. Those tissue samples will be evaluated for CYP27B1, CYP24A1, and VDR gene and protein expression using quantitative PCR and immunohistochemical assays.Objective 2: Determine the influence of vitamin D signaling in bovine and porcine immune responses. Other than upregulation of nitric oxide and RANTES production in bovine macrophages, the effects of vitamin D signaling on immunity of pigs and cattle is unknown. This objective seeks to identify the effects of vitamin D signaling on innate and adaptive immunity in vitro, and to develop a model to study the effects of vitamin D on disease resistance in vivo. Experiment 2.1: Identify genes regulated by vitamin D signaling in bovine and porcine immune cells. To identify the genes regulated in pigs and cattle LPS-stimulated and non-stimulated monocytes or epithelial cells will be treated with 0 or 10 nM 1,25(OH)2D3 for 6 and 24 h. RNA sequencing will then be performed on the mRNA isolated from the cells, and bioinformatics analysis will be performed to identify specific genes and pathways affected by 1,25(OH)2D3. RNA sequencing results from bovine and porcine immune cells will be validated with real-time PCR and appropriate functional assays.Experiment 2.2: Effects of vitamin D signaling on antigen-specific T cell responses of pigs. In cattle, humans, and mice, 1,25(OH)2D3 inhibits T cell proliferation and IFN-gamma and IL-17 production in vitro. To determine if 1,25(OH)2D3 that is produced by macrophages does the same in pigs, peripheral blood mononuclear cells (PBMCs; consist of macrophages, T cells and B cells) will be collected from H1N1-vaccinated and control and pigs. The PBMCs will be stimulated in culture with UV-killed H1N1 with and without 25(OH)D3. At the end of the culture period, the PBMCs will be labeled with fluorescent-conjugated antibodies specific for macrophages (CD14), T helper cells (CD4), and B cells (CD19), sorted via FACS according to cell type and evaluated for CYP27B1, VDR, and CYP24A1 gene expression to identify which cell populations are capable of producing and responding to 1,25(OH)2D3 in response to antigen stimulation. Effects of 25(OH)D3 on T cell proliferation and antigen-specific responses of each cell type in the PBMC cultures also will be evaluated to determine if 1,25(OH)2D3 produced in the PBMC cultures regulates antigen-specific immune responses of pigs.Experiment 2.3: Identify a method to precisely manipulate and maintain serum 25(OH)D3 concentrations in pigs. Considerable debate exists over the serum concentrations of 25(OH)D3 needed to support proper immunity. The ability to precisely and steadily control 25(OH)D3 concentrations in a relevant animal model of human immunity and vitamin D physiology needs to be developed in order to test the hypothesis that proper immune function relies on sufficient 25(OH)D3 availability. This experiments seek to identify the dosage amounts and intervals of vitamin D3 or 25(OH)D3 administration that can precisely maintain serum 25(OH)D3 at concentrations that are considered deficient (<10 ng/mL), insufficient (25 ng/mL), sufficient (50 ng/mL), high (75 ng/mL) and upper limit (100 ng/mL) for the human population, and are typical concentrations of swine housed outdoors (high) or from modern swine operations (deficient). Newly weaned pigs fed a vitamin D deficient diet and housed indoors at the UF swine unit will be divided into three groups (4/group) and administered the following treatments: 1) no supplemental vitamin D, 2) 10 μg/kg BW vitamin D3/week subcutaneously, or 3) 10 μg/kg BW 25(OH)D3/week subcutaneously. The treatments will start at weaning and continue for six weeks. Pigs will be weighed weekly and serum samples will be collected on the day of administration and again one day later and be analyzed for 25(OH)D (ELISA), 1,25(OH)2D (RIA), calcium and phosphorous (atomic absorption) concentrations. The study will be repeated as necessary to make adjustments and to demonstrate reproducibility from one experiment to the next.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Beef and Dairy farmers, nutritionists, veterinarians, scientists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Additional funding provided by SE milk check-off and allied industries to perform the objectives. This has led to opprotunites of international collaborations and training of three graduate students. How have the results been disseminated to communities of interest?Results have been reported to beef producers (Progressive Cattlemen, Forida Cattlemen Journal), dairy producers (Progressive Dairymen, FL dairy production conference), international scientific meetings, and nutrition meetings. What do you plan to do during the next reporting period to accomplish the goals?Continue ongoing studies on effects of dietary vitamin D on immunity and health.

Impacts
What was accomplished under these goals? Objective 1: We discovered 1) lipopolysaccharide induced mastitis, representative of coliform mastitis, stimulate vitamin D metabilism in macrophages and neutrophils of the mammary gland, 2) the active metabolite of vitamin D, calcitriol, stimulates expression of beta defensin and nitric oxide responses in the mammary glands of lactating cows, and 3) Th1 and Th2 cytokines have contrasting effects on vitamin D signaling in the bovine immune system. Objective 2: We surveyed beef and dairy cattle populations at various locations in the US. The survey revealed high risk of vitamin D deficiency in newborn beef and dairy calves. Feeding dairy cows 25-hydroxyvitamin D3 to elevate vitamin D status above normal decreased severity of mastitis compared with control cows. Ongoing projects are investigating the effects of supplemental vitamin D on health and performance of cattle.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Nelson, C.D., Powell, J.L., Price, D.M., Hersom, M.J., Yelich, J.V., Drewnoski, M.E., Bird, S.L., and G.A. Bridges. 2016. Assessment of serum 25-hydroxyvitamin D concentrations of beef cows and calves across seasons and geographical locations. J. Anim. Sci., 94:3958-3965.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Nelson, C. D., Lippolis, J.D., Reinhardt, T.A., Sacco, R.E., Powell, J.L., Drewnoski, M.E. ONeil, M., Beitz, D.C., and W.P. Weiss. 2016. Vitamin D status of dairy cattle: Outcomes of current practices in the dairy industry. J. Dairy Sci. 99:1-11.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Nutritionists, veterinarians, dairy producers, beef producers, allied industries. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Additional funding provided by SE milk check-off and allied industries to perform the objectives. This has led to opprotunites of international collaborations and training of three graduate students. How have the results been disseminated to communities of interest?Results have been reported to beef producers (Progressive Cattlemen, Forida Cattlemen Journal), dairy producers (Progressive Dairymen, FL dairy production conference), international scientific meetings, and nutrition meetings. What do you plan to do during the next reporting period to accomplish the goals?Continue ongoing studies on effects of dietary vitamin D on immunity and health.

Impacts
What was accomplished under these goals? Objective 1: We discovered 1) lipopolysaccharide induced mastitis, representative of coliform mastitis, stimulate vitamin D metabilism in macrophages and neutrophils of the mammary gland, 2) the active metabolite of vitamin D, calcitriol, stimulates expression of beta defensin and nitric oxide responses in the mammary glands of lactating cows, and 3) Th1 and Th2 cytokines have contrasting effects on vitamin D signaling in the bovine immune system. Objective 2: We surveyed beef and dairy cattle populations at various locations in the US. The survey revealed high risk of vitamin D deficiency in newborn beef and dairy calves. Feeding dairy cows 25-hydroxyvitamin D3 to elevate vitamin D status above normal decreased severity of mastitis compared with control cows. Ongoing projects are investigating the effects of supplemental vitamin D on health and performance of cattle.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Merriman, K.E., Kweh, M.F., Powell, J.L., Lippolis, J.D., and C.D. Nelson. 2015. Multiple ?-defensin genes are upregulated by the vitamin D pathway in cattle. Journal of Steroid Biochemistry and Molecular Biology. 154: 120-129.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Nelson, C.D., Powell, J.L., Price, D.M., Hersom, M.J., Yelich, J.V., Drewnoski, M.E., Bird, S.L., and G.A. Bridges. 2016. Assessment of serum 25-hydroxyvitamin D concentrations of beef cows and calves across seasons and geographical locations. J. Anim. Sci., 94:3958-3965.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Nelson, C. D., Lippolis, J.D., Reinhardt, T.A., Sacco, R.E., Powell, J.L., Drewnoski, M.E. ONeil, M., Beitz, D.C., and W.P. Weiss. 2016. Vitamin D status of dairy cattle: Outcomes of current practices in the dairy industry. J. Dairy Sci. 99:1-11.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Merriman, K.E., Poindexter, M.B., Kweh, M.F., Santos, J.E.P., and C.D. Nelson. 2017. Intramammary 1,25-dihydroxyvitamin D3 treatment increases expression of host-defense genes in mammary immune cells of lactating dairy cattle. J. Steroid Biochem. Mol. Biol. doi: 10.1016/j.jsbmb.2017.02.006.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Nelson, C.D., Merriman, K.E., Poindexter, M.B., Kweh, M.F., Blakely, L. Targeting antimicrobial defenses of the udder through an intrinsic cellular pathway. J. Dairy Sci.

Progress 02/20/14 to 09/30/14

Outputs
Target Audience: Animal Scientists, Veterinarians, Nutritionists, Dairy Producers, Beef Producers, Swine Producers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project has provided research opportunities for three graduate students, and five undergraduate students. The graduate students have presented their work at regional and national scientific meetings. The project has provided over 300 hours of undergraduate research experience. The undergraduates have fulfilled their honor's thesis and internship research requirements for their degrees. How have the results been disseminated to communities of interest? Outcomes of our research efforts have been presented to Dairy Scientists (2014 ASAS/ADSA Joint Annual Meetings, Kansas City MO, >2,000 attendees), Dairy Producers (2014 FL Dairy Production Conference and 2014 S Florida Dairy Extension meeting, 20 attendees), Veterinarians and Nutritionists (2014 FL Ruminant Nutrition Symposium, 200 attendees) What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? IMPACT: Our research has indicated vitamin D has a critical role in activation of cattle and swine immune systems. Through our research efforts we also have observed widespread vitamin D defficiency among US swine and cattle populations. Our data indicates that inadequate vitamin D impairs the ability of livestock to defend against viral and bacterial pathogens that result in wide prevalence of bacterial and viral diseases in the US swine and cattle populations. Specific accomplishements: Objective 1: Identify effects of host and pathogen factors on vitamin D metablism in the immune system. -Our research team has determined that activation of the swine immune system initiates production of the active vitamin D hormone. -We have identified age and gender of cattle as key determinants of vitamin D metabolism in the immun system. Objective 2: Determine the influence of vitamin D signaling on immune responses. - We have identified several importatn host defense genes that are induced by the vitamin D hormone. - We have developed a model in calves to study the effects of vitamin D status on immunity.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Nelson C.D., and K.E. Merriman. 2014. Vitamin D Metabolism in Dairy Cattle and Implications for Dietary Requirements. 25th Annual Florida Ruminant Nutrition Symposium. 78-91. http://dairy.ifas.ufl.edu/rns/2014/nelson.pdf
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Nelson C.D., Merriman K.E., and J.D. Lippolis. 2014. Vitamin D signaling enhances expression of antibacterial beta defensin genes in bovine monocytes. American Dairy Science Association - American Society of Animal Science Joint Annual Meeting,Kansas City, MO
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Merriman K.E., and C.D. Nelson. 2014. Evaluation of the effects of vitamin D and toll-like receptor signaling pathways on expression of antibacterial ?-defensin genes in bovine neutrophils and mammary epithelial cells. American Dairy Science Association - American Society of Animal Science Joint Annual Meeting,Kansas City, MO.
• Type: Other Status: Published Year Published: 2014 Citation: Nelson C.D. 2014. Why cattle need vitamin D. Progressive Dairyman. Issue 14: 62-63. http://www.progressivedairy.com/